This invention relates generally to preparation of blood serum samples for biochemical analyses performed by photometric methods.
When performing biochemical analyses on blood serum or other body fluids, difficulties will be encountered time to time due to the presence of substances abnormally present in the serum, these substances producing direct absorption of light in a colorimetric measurement which is additive to the color intensity factors of the specific chemical reaction being observed.
Serum not only contains triglycerides which are held in solution conjugated with protein such as lipo-protein, principally beta-lipo-protein. As the proportion of protein in lipo-protein molecules decreases, the solubility of the molecule decreases. When the protein content reaches about 5 percent, the lipo-protein exists as finely emulsified droplets having a diameter greater than 0.5 microns and called chylomicra. These are visible and responsible for a serum turbidity called lipemia. Lipemia usually appears above a total serum lipid level of 500 to 1,000 milligrams per deciliter, but serum may remain optically clear at a liquid level as high as 4400 per milligrams per deciliter. Even clear serum or plasma may develop turbidity on standing, possibly as a result of aggregation of the lipo-protein molecules. The appearance of lipemia normally occurs after a meal containing fat. The sample turbidity due to chylomicrons, is common and is a source of error due to light scattering or light absorption by the fatty particles present in the specimen. This type of turbidity has been found to be extremely difficult to remove without subjecting the sample to a fat-extraction process which causes alteration or damage to other biochemical constituents which may be of interest.
It has been observed that when nonlipemic blood is subjected to a centrifugal field, a white zone appears at the air-plasma interface and when the centrifugal field is removed, the white zone disappears. The field strength range through which this phenomenon occurs has been found to be RCF 500 to 1000 where RCF is the relative centrifugal force. When lipemic blood was observed under the same conditions, the white zone was very pronounced. Further, the plasma between it and the red cell layer became quite clear. When the centrifugal field was removed, there was substantial redistribution of the white zone into the plasma although in the case of lipemic blood there remained a residual concentration within the white zone. Accordingly, it has been found that the removal of chylomicrons from lipemic blood by simple centrifugation is considerably difficult if not impossible.
Another suggestion for declarification would be the utilization of specific solvents added to the sample so as to extract the microscopic fat particles in known manner. A typical solvent would be ether mixed with the serum and then permitted to separate by gravity. As stated above, all the polar molecules in the serum as well as the chylomicrons would be exposed to the solvent action of the ether, hence such polar molecules as the sterols, e.g., cholesterol, would be affected. Thus, there would be a substantial disturbance of the relative concentration of the lipid-soluble components with deleterious results on the sample for further testing.
Accordingly, the invention provides a method for clarifying the lipemic blood serum samples having turbidity due to chylomicrons without subjecting the serum samples to the disadvantages encountered using either centrifugation alone or extraction with specific solvents.